Nucleic Acid Detection Ultrasensitive Fluorescent Gel Stains and Quantitation Reagents ® Molecular Probes has developed nucleic acid stains that, in addition to having high affinities for nucleic acids, also exhibit very high fluorescence enhancements upon binding (>300-fold) compared to conventional stains such as ethidium bromide and Hoechst 33258. Our dyes also have high extinction coefficients and quantum yields, result- ing in extremely strong fluorescence signals. All of these properties combine to make our nucleic acid stains the easiest to use, most reliable and highest-sensitivity dyes for gel staining and solution quantitation. SYBR dyes: the most sensitive gel stains SYBR nucleic acid gel stains are the most sensitive stains available for nucleic acid detection in gels, allowing you to use a fluorescent dye to visualize bands that previously could only be detected using labor-intensive silver staining or radioactive labeling techniques. Advantages Highly sensitive. Up to 25-fold more sensitive than ethidium bromide, SYBR dyes provide sensitivity rivaling that of silver staining. Easy to use. One-step staining and detection does not require destaining or washing. Compatible with molecular biology techniques. Stained nucleic acids can be used for Northern or Southern blotting and in enzymatic reactions such as ligations, restriction digests, amplification reactions and in vitro transcription. If desired, the stain can be removed from extracted bands by a simple ethanol precipitation. Figure 1. Direct visualization of SSCP in exon 1 of human K-ras using SYBR Gold nucleic acid gel stain. Lane 1 con- tains wild-type DNA and lanes 2–4 contain DNA from vari- ous adenocarcinoma samples with mutant alleles. Image contributed by Valerie DeGroff and Chris Weghorst, Ohio State University. Figure 2. Negative image of a DGGE gel stained with SYBR Green II dye. The different migration patterns of 5S rRNA from Leptospirillum ferroxidans BU-1 strain, Thiobacillus thiooxidans ATCC 8085 strain and an iron- oxidizing heterotrophic bacterium, SLC2, can be seen in the left half of the gel. Image contributed by Daphne Stoner, Idaho National Engineering Laboratory. SYBR Gold nucleic acid gel stain The most sensitive and versatile fluorescent stain for use with UV transilluminators This newest SYBR dye outperforms ethidium bromide in any gel system, including agarose and polyacrylamide gels, native gels, formaldehyde gels, glyoxal gels and urea gels.1 The stain penetrates thick gels easily for fast and even staining. It is the most sensitive stain for dsDNA, ssDNA and RNA using a standard 300 nm UV transillumi- nator, enabling you to obtain high sensitivity without using expensive laser scanners. Applications Northern blotting Stained RNA transfers easily onto nitrocellulose or nylon membranes by standard blotting methods, without loss of the sample. The stain washes off of the RNA during the prehybridization step and does not interfere with hybridization. SSCP analysis SYBR Gold stain is ideal for “cold” single-strand conformation polymorphism (SSCP) analysis, eliminating the need for radioactivity in this gel-based allele detection assay (Figure 1). PCR-based assays As sensitive as silver stains, but much easier to use,1 SYBR Gold stain provides the ideal detection method for PCR-based gel assays requiring high sensitivity, such as the telomeric repeat amplification protocol 2 (TRAP). SYBR Green II nucleic acid gel stain A high-sensitivity dye designed for staining RNA in gels SYBR Green II stain shows especially good sensitivity for RNA, while also staining dsDNA and ssDNA. The ideal dye for use with laser scanning instruments, SYBR Green II stain exhibits very low background fluorescence in the gel and has spectral characteristics that match common light sources and filter sets. Applications Northern blotting SYBR Green II stain shows high-sensitivity RNA staining in formaldehyde gels without the need for destaining. Staining with SYBR Green II dye does not interfere with subsequent RNA transfer or blotting.3 Staining the gel prior to blotting provides a means of normalizing the hybridization signals.4 DGGE analysis This stain makes it possible to perform a very sensitive assay that characterizes species in a mixed microbial population. The assay is based on the migration of bacterial 5S rRNA during denaturing gradient gel electrophore- sis 5 (DGGE, Figure 2). SSCP analysis SYBR Green II stain provides a simple method for high-sensitivity, nonradioactive single-strand conformation polymorphism (SSCP) analysis.6–8 References 1. Anal Biochem 268, 278 (1999); 2. Mol Pathol 51, 342 (1998); 3. J Chinese Biochem Soc 32, 1 (1995); 4. BioTechniques 26, 46 (1999); 5. Appl Environ Microbiol 62, 1969 (1996); 6. Diagnostic Mol Pathol 5, 260 (1996); 7. Anal Biochem 236, 373 (1996); 8. Proc Natl Acad Sci USA 94, 10745 (1997). SYBR Green I nucleic acid gel stain A dsDNA-selective dye with exceptionally low background The well-established SYBR Green I stain preferentially stains dsDNA, making it especially useful for assays where the presence of contaminating RNA or ssDNA might otherwise obscure the results. With exceptionally low back- ground fluorescence and spectral characteristics that closely match light sources and filter sets in existing instru- ments, SYBR Green I stain is ideal for use with laser scanners. Applications Complex samples Preferential dsDNA staining makes it easy to detect dsDNA patterns, such as apoptosis ladders, even in crude extracts (Figure 3). DNA typing SYBR Green I stain shows much higher DNA staining sensitivity than does ethidium bromide and can replace silver staining or radioisotope labeling in gel-based DNA-typing assays.1–6 PCR-based assays The stain also improves the sensitivity of other gel-based PCR assays, such as viral detection assays 7 and the telomeric repeat amplification protocol 8 (TRAP). Increased sensitivity means improved accuracy in competitive reverse transcription-PCR (RT-PCR) because fewer cycles are required.9–11 Band-shift assays SYBR Green I stain can replace radioactivity for detection of protein-bound and unbound DNA in band-shift assays 12,13 (Figure 4). DNA damage assays SYBR Green I stain makes assays for DNA damage easier, safer and more sensitive. It has been used to replace tritium-labeling of DNA in a pulsed field gel electrophoresis (PFGE) assay 14 and to increase the sensitivity of the popular comet assay.15 References 1. BioTechniques 19, 223 (1995); 2. BioTechniques 22, 976 (1997); 3. Nature Biotech 16, 91 (1998); 4. Biochim Biophys Acta 1360, 193 (1997); 5. Mol Cell Probes 9, 145 (1995); 6. J Forensic Sci 44, 87 (1999); 7. J Virol Meth 55, 153 (1995); 8. J Biol Chem 274, 7264 (1999); 9. J Biol Chem 274, 21893 (1999); 10. PCR Meth Appl 4, 234 (1995); 11. BioTechniques 22, 1107 (1997); 12. FASEB J 10, A1128, abstract #751 (1996); 13. J Biol Chem 274, 27287 (1999); 14. Nucleic Acids Res 25, 2945 (1997); 15. www.kineticimaging.com/komet.htm. Figure 4. Band-shifts detected with SYBR Green I stain. Samples containing 50 ng of a 208 bp DNA fragment and varying amounts of a mutant enzyme (EcoRI/Gln 111) were electrophoresed through a native polyacrylamide gel then stained with SYBR Green I stain. Lanes 1 and 10 contain size markers; lanes 2 through 9 contain 0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 0 µM EcoRI/Gln 111. Figure 3. Detection of DNA fragments in apoptotic cells. DNA extracts from HL-60 cells treated with the apoptosis- inducing compound camptothecin were separated on an agarose gel then stained with SYBR Green I nucleic acid gel stain. The 200 to 5000 bp DNA fragments characteristic of apoptotic cells appear as “ladders.” Cell preparations were gifts of Zbigniew Darzynkiewicz, Cancer Research Institute, New York Medical College. SYBR Green/Gold photographic filter Documenting a fluorescent image on film or with a CCD camera allows the image to be integrated over time, so you will see fluorescence signals not detectable with the human eye alone. To obtain the highest sensitivity with the SYBR nucleic acid gel stains using a UV transilluminator and Polaroid black-and-white print film, we recommend the use of the SYBR Green/Gold photographic filter. This simple and inexpensive 3 × 3–inch gelatin filter blocks out background UV light while allowing the maximum amount of SYBR stain fluorescent signal to reach the camera. For CCD cameras, laser scanners or cameras requiring screw-in glass filters, please contact the instrument manufacturer for the appropriate filters. PicoGreen, RiboGreen and OliGreen dyes: the most sensitive solution quantitation reagents Molecular Probes’ PicoGreen, RiboGreen and OliGreen quantitation reagents show very high fluorescence enhancements upon binding to nucleic acids. This characteristic provides a simple, high-sensitivity method for quantitating nucleic acids in solution. The high sensitivity means you save more of your precious samples for research. The one-step assays are easily adaptable for use in high-throughput settings. Advantages Sensitive and accurate. These fluorescent dyes are orders-of-magnitude more sensitive than UV absorbance (A260) readings or assays using Hoechst 33258. And in contrast to A260 measurements, nucleic acids can be quantitated without interference from proteins or free nucleotides. Fast and easy. These one-step assays require only a five-minute incubation and are easily adapted to robotic high- throughput quantitation. Compatible with most instruments. Fluorescent signals match the excitation sources and optical filters